Somatic cells have a limited lifespan and gradually slow in growth and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation.
The life cycle of human papillomavirus (HPV) is best studied in primary human keratinocytes, the natural host cells of HPV. Papillomaviruses infect the mitotically active cells of the basal layer of the epithelium, but viral progeny are only produced when these infected precursor cells differentiate. Papillomavirus infections are persistent and the viral genome is maintained in the continuously dividing basal cells for long time periods. The HPV genome can be transfected into isolated keratinocytes where it becomes established as an extrachromosomally replicating element. These infected cells can be induced to differentiate and stratify and support the productive cycle of HPVs (Frattini et al., Proc. Natl. Acad. Sci, USA 93:3062-3067, 1996; Meyers et al., Science 257:971-973, 1992).
A subset of about 15 papillomaviruses from the alpha genera is associated with cancer, primarily of the uterine cervix (Smith et al., Int. J. Cancer 121:621-632, 2007). Almost 100% of cervical carcinomas contain a “high-risk” HPV. These “high-risk” viruses are also associated with a subset of head and neck cancers (Gillison and Shah, Curr. Opin. Oncol. 13:183-188, 2001). These cancer-associated HPVs are also able to immortalize primary human keratinocytes in culture (Hawley-Nelson et al., EMBO J. 8:3905-3910, 1989; Munger et al., J. Virol. 63:4417-4421, 1989). Under the appropriate culture conditions, the viral genome is maintained as an extrachromosomal element and the functions of the viral E6 and E7 oncoproteins provide a selective growth advantage for these cells (Goodwin et al., Proc. Natl. Acad. Sci. USA 97: 10978-10983, 2000). These cells can be further cultured as an organotypic raft where progeny virus can be produced (Frattini et al., Proc. Natl. Acad. Sci, USA 93:3062-3067, 1996).
Much less is known about the “low-risk” HPVs that are not associated with malignant carcinomas. The genomes of these viruses can be introduced into primary cells, but the E6 and E7 proteins do not provide a growth advantage to the cells which will often senesce before extensive studies can be carried out or before they can be cultured in an organotypic raft. These “low-risk” viruses are, however, the causative agents of a wide range of benign, proliferative lesions that can cause intractable disease. These viruses can be studied in immortalized keratinocyte cell lines, but these lines have been shown to have genetic abnormalities that could interfere with functional analyses of the virus (Lehman et al., Carcinogenesis 14:833-839, 1993). Thus, a need exists to increase the proliferative capacity of human primary keratinocytes and to develop an efficient means to induce immortalization of these cells. Such methods are desirable not only for studies of HPV replication, but for a variety of therapeutic purposes.